Insight into post-transcriptional regulatory mechanisms of bacterial gene expression will be sought through the study of a novel paradigm in global regulation, the carbon storage regulatory system (Csr) of Escherichia coli. Csr includes an RNA-binding protein, CsrA, that regulates translation and/or modulates the stability of specific mRNAs, and two small non-coding RNA molecules, CsrB and CsrC, which antagonize CsrA activity. It is hypothesized that CsrB and CsrC interfere with CsrA-mediated regulation by competing with regulated mRNAs for CsrA binding. In E. coli, CsrA affects metabolism, physiology and multicellular behavior on a broad scale, repressing certain genes expressed during the transition from the exponential to stationary phase of growth and activating various genes expressed during exponential phase. CsrA homologues are widely-distributed among eubacteria and regulate the expression of virulence factors in both plant and animal pathogens. Thus, the proposed studies will also provide fundamental understanding of the regulation of bacterial physiology and pathogenesis, and may suggest novel therapeutic approaches for bacterial infections. Specific aims of this proposal are: 1) To further elucidate the molecular mechanisms responsible for CsrA-mediated activation or inhibition of gene expression. This will include determination of the sequence and structural requirements for mRNA recognition and the mechanism by which CsrA-mediated translational inhibition leads to degradation of target transcripts. 2) We will assess the molecular mechanisms and regulatory roles of small noncoding RNAs in CsrA antagonism. The isolated CsrA-RNP complex will be characterized to determine the RNA segments involved in CsrA binding. A novel RNA that complexes with CsrA will be characterized. 3) We will delineate the genetic, physiological and environmental factors to which the Csr system responds. We will examine the physiological and molecular basis by which flux through the cysteine biosynthesis pathway regulates csrA gene expression. The long-range basic objectives of these studies are to fully understand the regulatory components and genetic circuitry, molecular mechanisms, and biological functions of the Csr system.